Nucleic Acids Research, 1991, Vol. 19, No. 5 1129-1137
© 1991
MOLECULAR BIOLOGY |
A segment of a plasmid gene required for conjugal transfer encodes a site-specific, single-strand DNA endonuclease and ligase
Department of Microbiology, University of Texas Austin, TX 78712, USA
* To whom correspondence should be addressed
Received August 27, 1990. Revised January 22, 1991. Accepted January 22, 1991.
The polypeptide encoded by a segment of a gene required for the conjugal mobilization of the broad host-range plasmid R1162 has been purified as a ß-galactosidase fusion protein. The hybrid protein binds specifically to a small, double-stranded DNA fragment containing the origin of transfer (oriT), and specifically cleaves orIT single-stranded DNA at the position cleaved during transfer. Only one of the two DNA strands is a substrate. A fraction of the digested DNA is resistant to 
exonuclease digestion, indicating that some molecules have protein covalently attached at the 5' end. After prolonged incubation with fusion protein, some of the cleaved molecules are religated. In vivo, M13 phage DNA containing two, directly-repeated copies of oriT recombine in cells containing the fusion protein. The single-stranded viral DNA forms are the probable substrates for the protein, the cleaved DNA being subsequently religated to form recombinant molecules. Cleavage of the DNA might be the reverse reaction of the ligatlon that normally takes place after conjugative transfer of a single, linear plasmid DNA strand.
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